Most newly transformed cancer cells are recognised and are eliminated by the immune surveillance. Drastic cancerous transformations grant immune-escape features and, the abilities to alter and hijack the immune system for cancer cell survival and growth. Here, we describe the multiparametric screening pipelines developed in our laboratory to identify cancer-driven biomarkers, starting in preclinical setting to train Machine Learning model that can predict the presence and the type of cancer. Our goal is to translate these workflows into the clinic.


Affiliations: Irradiation Immunity Interaction (I3) Laboratory, Division of Genome Sciences and Cancer, John Curtin School of Medical Research, Australian National University, Australia.

The most common imaging examinations for diagnosing pulmonary embolism are CTPA and VQ. However, a previous lack of high-quality data and analysis made it challenging to provide an accurate comparison of maternal and foetal risk of radiation-induced cancer between each imaging technique.

 

To estimate the cancer risk from maternal and foetal radiation doses associated with CTPA or VQ examinations.

 Methods

Dosimetric data was determined for 274 pregnant patients who received CTPA and/or VQ examinations at CHS between 2015 and 2022. The maternal incident and mortality radiation risks were estimated using models provided by the Biologic Effects of Ionizing Radiation (BEIR) VII report. The BEIR models account for age at exposure, time following exposure, and organ dose. Input data included median and quartile values of determined critical organ dose estimates and patient age. Risk to the foetus was determined using standard methods.

 Results

Estimated total maternal cancer incidence was 27 and 26 cases per 100,000 persons for CTPA and VQ examinations respectively, with mortality estimates of 11 and 15 deaths respectively. Cancer incidence was evenly divided between lung and breast cancer for CTPA with 80% of cancer cases for VQ being in the lung. The median foetal doses were 0.03 mSv for CTPA, and 0.29 mSv for VQ, yielding 0.5 cases of radiation-induced childhood cancers from CTPA and 4.4 cases per 100,000 persons for VQ.
 
 

Maternal cancer incidence is remarkably similar for each examination.
 
 

Knowledge of the potential long-term risks following radiation exposure from diagnostic imaging procedures is essential in the practices of justification and optimisation.

Donald McLean1, Olivia Delfino1, Marie Vozzo1
 
 

1.     Medical Physics and Radiation Engineering, Canberra Health Services, Garran, ACT, 2605

NODk mice is protected from T1D due to congenic replacement of Idd1 MHC locus with an autoimmune diabetes-resistant H2k locus. However, male NODk mice fed a western-diet (WD) develop a type 2 diabetes (T2D) like phenotype characterised by weight gain, dyslipidaemia, hyperinsulinaemia, and severe hyperglycaemia.

To determine if time-restricted nutrient supply, via intermittent fasting (IF) regimen, can prevent development of hyperinsulinaemia, excessive weight gain, glucose intolerance and reduce incidence of diabetes in WD fed male NODk mice.

Methods: Male NODk mice were randomised to chow diet (CD), WD or WD with IF (WD+IF) from 6 weeks until 10 or 30 weeks of age. Their metabolic characteristics were assessed, including intraperitoneal glucose tolerance testing (ipGTT) after 3 and 17 weeks on diet, and incidence of diabetes after 24 weeks on diet.

IF attenuated WD-induced body weight (24 weeks on diet (meanSEM); 38.3±0.6, 51.4±0.7, 47.0±0.5 g; CD, WD, WD+IF, respectively; diet effect p<0.0001), prevented WD-induced glucose intolerance (ipGTT glucose AUCGlu; 1016±45, 2230±127, 1433±56, mM*min; diet effect p<0.0001); and prevented WD-induced hypertriglyceridaemia (124±25, 432±46, 216±24 mg/dL; diet effect p<0.0001). WD-induced hyperinsulinaemia (fed-state; 22.7±1.4 vs 14.3±0.5 ng/mL; WD, WD+IF; p<0.05), and during ipGTT (ipGTT insulin AUCIns after 17 weeks on diet; 35425 vs 25129 ng/ml*min; WD, WD+IF; p<0.05) was attenuated. IF prevented WD-induced diabetes after 24 weeks (diabetes in 0%, 81%, 0%, respectively; p<0.0001).

IF attenuated WD-induced weight gain, hyperinsulinaemia, hypertriglyceridaemia, glucose intolerance, and prevented diabetes in WD fed NODk mice.

 

These results suggest IF to be a promising nutrient deprivation approach for prevention and treatment of T2D


Muhammad Shamoon1,2, Jane Dahlstrom3, Viviane Delghingaro-Augusto4, Christopher Nolan1,2,5
 
 

1.     School of Medicine and Psychology, The Australian National University, Acton, ACT 2600 

2.     Division of Immunology and Infectious Diseases, John Curtin School of Medical Research, The Australian National University, ACT, 2600

3.     Department of Anatomical Pathology, ACT Pathology, Canberra Health Services, Garran, ACT 2605

4.     Division of Genome Science and Cancer, John Curtin School of Medical Research, Australian National University, Canberra ACT 2600, Australia

5.     Endocrinology and Diabetes, Canberra Health Services, Garran, ACT 2605

Cells communicate by transferring molecular cargo using nanosized delivery vehicles called extracellular vesicles (EV). As we age, there is loss of EV-mediated communication, leading to the progression of retinal degenerations such as Age-related Macular Degeneration (AMD). We hypothesise that if we can supplement the retina with cargo lost during degeneration, we can restore cellular communication and slow the progression of AMD. 

We propose to use autologous-sourced EV from red blood cells (RBC-EV) as delivery vehicles of these essential retinal EV cargo and develop a novel gene therapy for AMD.

EV were isolated from RBCs using differential ultracentrifugation and characterised using nanoparticle tracking analysis, electron microscopy and western blotting . RBC-EV were labelled with an RNA dye and transfected in-vitro into 4 retinal cell lines. The safety and uptake efficiency was analysed using Fluorescence Live cell imaging. In-vivo safety and uptake efficiency was investigated using functional and histological analysis.

Labelled RBC-EV were safely and efficiently uptaken by all retinal cell types with upto 90% transfection efficiency within 24 hours. In-vivo, intravitreal injection of RBC-EV showed significant protection in murine model of AMD as recorded in both functional readouts and histological analysis. Further, labelled-RBC EV were uptaken by cells in ganglion cell layer, inner nuclear layer and external limiting membrane of the retina within 6 hours of injection.

This work not only supports use of RBC-EV as therapeutic delivery agents but shows their efficacy in native state as therapeutics.

RBC EV loaded with healthy retinal cargo is a novel and promising gene therapy option for AMD.


Rakshanya Sekar, Yvette Wooff, Adrian Cioanca, Riccardo Natoli
 
 

1.     JCSMR, The Australian National University, Acton, ACT, 2601 

Waldenström Macroglobulinaemia (WM) is a B-cell lymphoma with clinical features including thrombocytopenia, IgM-mediated hyperviscosity, bleeding and bruising. Treatment of WM patients with Bruton’s tyrosine kinase inhibitors (BTKis) may exacerbate bleeding risk. Abnormal haemostasis may result from platelet dysfunction and altered coagulation, however the extent of dysregulation of these processes in WM patients is unknown.

 

To evaluate haemostatic and platelet dysfunction in WM patients. 

 

Platelet receptor levels in samples from 19 clinically annotated WM patients or healthy donors (HDs) were measured by flow cytometry. Thrombin generation in plasma ± platelets was measured by fluorescence resonance energy transfer assay. Whole blood clotting potential was evaluated by rotational thromboelastometry (ROTEM). Levels of plasma soluble GPVI (sGPVI) and serum thrombopoietin (TPO) were measured by enzyme-linked immunosorbent assays. 

 

WM platelets had reduced levels of GPIbα (p=0.0153), GPVI (p=0.0347) and reticulation (p=0.0005), and increased overall sialylation (p=0.0079) and tetraspanin CD9 levels (p<0.0001). WM plasma displayed significantly reduced thrombin generation potential (p=0.0408) and WM platelets contributed less to thrombus initiation (p=0.0208) and rate (p<0.0001) by ROTEM, but displayed normal responses to agonists, except in the presence of BTKis. Plasma sGPVI were within normal ranges and TPO levels were increased (p<0.0001).

 

The impaired haemostatic potential in WM samples was independent of BTKi therapy. Reduced platelet GPIbα and GPVI may arise from altered megakaryocyte maturation and thrombopoiesis, associated with bone marrow malignancy. Monitoring platelet and coagulation properties may help stratify WM patients for bleeding risk.


Simone A Brysland, Philip J Crispin, Dipti Talaulikar, Elizabeth E Gardiner
 
 

1.     Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, The Australian National University, Canberra, 2601, ACT. 

Department of Haematology, Canberra Health Services, Garran, ACT 2605 

To facilitate precision medicine, we require a detailed understanding of the biological pathways that are affected. The Canberra Clinical Phenomics Service will deliver cellular phenotyping and sophisticated analysis of lymphocytes in patient blood samples. 

 

We aimed to create a flow cytometry panel for the purpose of deep phenotyping human lymphocyte subsets. To expand flow cytometry tests beyond what is already available, we utilised a spectral flow cytometer, which permits more parameters to be investigated at once compared to conventional flow cytometry.

 

Ficoll-separated PBMCs were stained with fluorescent antibodies and analysed on a Cytek Northern Lights 3-laser spectral cytometer.

 

Through careful panel design, panel testing, titration and method optimisation, we generated a 30-colour lymphocyte panel. We can consistently detect populations including T cells (alpha-beta and gamma-delta); CD4+ and CD8+ naïve, TEMRA, central and effector memory; cTfh cells, Tregs and Tfr cells; CD57+ exhausted T cells and B cells; naïve, T1 and T2 transitional; switched and unswitched memory; IgG+ and IgA+ memory; Ig-kappa and Ig-lambda chain expression, Bregs, atypical CD11c+ B cells; activated B cells, plasmablasts and plasma cells.

 

We have developed a panel of 30 markers that can be simultaneously analysed, allowing the characterisation of 62 T cell and B cell subpopulations in a single sample. 

 

Offering such a broad panel maximises both the amount of data generated and the number of research and diagnostic areas to which it can be applied. Our long-term aim is for this panel to be accredited by the TGA and NATA as a diagnostic test.


Ainsley R Davies1, Kristy Kwong1, Ann-Maree Hatch1, Philip Wu1, Minnal Abbasi1, Koula Diamand1, Fei-Ju Li1, Stuart Read1, Harpreet Vohra2, Michael Devoy2, Euan McNaughton1, Katrina Randall1,3
 
 

1.     Canberra Clinical Phenomics Service, John Curtin School of Medical Research, The Australian National University, Acton, ACT, 2000

2.     Flow Cytometry Facility, John Curtin School of Medical Research, The Australian National University, Acton, ACT, 2000

3.     Department of Clinical Immunology, Canberra Health Services, Garran, ACT, 2605